Without limiting the scope of the invention, its background is described in connection with multiplex probes.
Current methods of protease activity detection involve fluorescence detection using selectively activated probes. Enzyme-linked immunoassay (ELISA) based methods for the detection of this enzyme take advantage of the sensitivity of fluorescence measurements. All of these techniques, however, involve multiple, time-consuming steps. The length of the process combined with the expense and the expertise involved in the setup/analysis of these techniques make them impractical for clinical practice.
One such system is taught in U.S. Pat. No. 8,609,329, issued to Barroso, et al., which is directed to a FRET-based assay for screening modulators of receptor cycling. Briefly, these inventors teach an automated FRET imaging of membrane-bound receptor/ligand complexes that is said to discriminate between a clustered organization of ligand/receptor complexes that occurs during the early endocytic stages following internalization and a random distribution characteristic of late stage disassociation of ligand from the receptor.
Another system is taught in U.S. Reissue Pat. No. RE42,325, to Wittwer, et al., which is directed to a homogeneous multiplex hybridization analysis by color and Tm. Briefly, this invention is said to provide methods and devices for analyzing sequence variations in nucleic acid samples that include multiple loci, each having two, three or more possible allelic sequences. The method is said to involve combining at least a first and second pair of oligonucleotide probes with the nucleic acid sample. The first pair of probes is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the first locus and the second pair is capable of hybridizing in proximity to each other within a segment of the nucleic acid sample comprising the second locus. The first member of each probe pair comprises a FRET donor and the second member comprises a FRET acceptor, the FRET acceptor of the first probe pair member having a different emission spectrum from the FRET acceptor of the second probe pair. Upon hybridization, the proximity of the first and second member of each probe pair is sufficient to allow fluorescence resonance energy transfer between the FRET donor and the FRET acceptor.